Various means are now known for fractionating substances such as proteins according to their charges and molecular weights by electrophoresis and isolating the obtained fractions, which include a fractionation capillary electrophoresis apparatus, a preparative liquid electrofocusing apparatus and a method in which electrical separation is conducted in a gel and thereafter a desired substance is extracted from the gel. However, the above apparatus and method have the following drawbacks.
The fractionation capillary electrophoresis apparatus is one comprising a linear capillary of 1 mm or less in diameter provided with electrodes at its both ends, the separative power (capability of fractionation) of which is extremely high. However, the isolation of a substance separated by electrofocusing is conducted at the end of the capillary, so that it is required to push out the separated substance from the capillary. This operation gives an unfavorable influence such as disturbance of the separation pattern, thereby bringing about the disadvantage of rendering fractionation with high accuracy unfeasible. Further, the diameter of the capillary is as small as 1 mm or less that the amount of the sample subjected to electrophoresis is so small as to bring about a drawback in that the apparatus is unsuitable for use in the step of electrophoresis requiring a large amount of sample in one operation as in the structural analysis of, for example, an amino acid sequence.
The preparative liquid electrofocusing apparatus has such a construction that electrofocusing is performed in a cylinder having its inside partitioned into 20 sub-sections with membranes. Thus, electrophoresis is performed in an electrophoresis pipe partitioned into multiple sub-sections by suitable membranes, so that the diffusion of the separated substance is suppressed even after the discontinuation of the electrification with the result that this apparatus is free from the above noted drawbacks of the conventional fractionation capillary electrophoresis apparatus. However, the number of sub-sections is only 20, so that the separative power of the apparatus is relatively low to thereby bring about a drawback in that fractionation with high accuracy cannot be ensured. Moreover, this apparatus has the problem that the adsorption of proteins by the membranes, and the membrane clogging attributed to isoelectric precipitation, are detrimental to the separative power of the apparatus.
The above method in which the desired substance is extracted from a gel specifically comprises performing electrophoresis with the use of a gel prepared from, for example, polyacrylamide as an electrophoresis solution, staining the sample after the electrophoresis to thereby discriminate the desired substance, cutting out a portion of the gel containing the desired substance, and extracting the desired substance from the cut out gel portion. A sample as large as several milligrams can be processed depending on the gel size, and the separative power of the apparatus is relatively high. However, the step of extracting the desired substance separated by electrophoresis from the cut out portion of the gel is required, thereby posing the problems of a reduction in the extraction efficiency and contamination with impurities originating in the gel.